The long-term goal of the proposed research is to understand and define the biochemical and structural aspects of HCV replicase. This would necessitate identification of the amino acid residues, as well as specific domains on the enzyme molecule involved in the process of copying the viral RNA genome. Recently solved three-dimensional crystal structures of HCV replicase in conjunction with molecular modeling have pinpointed a number of amino acid residues suspected to be involved in specific steps of the enzymatic reaction. These residues will be targeted for site directed mutagenesis and the resulting mutant enzymes will be characterized with respect to their biochemical properties in order to elucidate their functional roles in the catalytic process. Mutagenesis studies along the putative RNA binding track will clarify the roles of these residues in the process of RNA replication. These studies will have direct application in defining the side chain orientation of a number of amino acid residues in the catalytic domain. Knowledge of the complete functional 3-D structure of HCV replicase will help in better understanding of the mechanisms of RNA dependent RNA synthesis and inhibitory action of anti HCV-replicase drugs. Availability of this information, in turn, will further facilitate development of new HCV-replicase directed agents of specific atomic dimensions that would act at specific structural elements of HCV-replicase. The following specific aims are proposed. Aim 1: Biochemical characterization of HCV replicase. Aim 2: To elucidate the catalytic roles of specific amino acid residues in the polymerase function. Aim 3: To determine protein-protein interactions between HCV replicase and other nonstructural viral proteins.